Sulfated polysaccharids for treatment or prevention of thromboses

ABSTRACT

The invention relates to new mixtures of sulfated oligosaccharides having the general structure of heparin constitutive oligosaccharides, having an average molecular mass of 6±0.6 kD, a polydispersiveness close to 1 and the capacity of inhibiting the production of thrombin. The invention also relates to their preparation and to pharmaceutical compositions containing them.

This is a continuation of application Ser. No. 08/137,137 filed on Oct.21, 1993, now abandoned, which is the U.S. national stage entry under 35U.S.C. § 371 of PCT/FR92/00352, filed on Apr. 21, 1992 published asWO92/18544, Oct. 29, 1992.

The present invention relates to the field of low-molecular weightpolysaccharides. More specifically, it relates to oligosaccharidecompositions possessing excellent pharmacological and antithromboticproperties.

Generally, antithrombotic treatments require the use of two maincategories of agents, namely anticoagulatory agents and antiplateletagents.

Antivitamin K compounds constitute a very important family among theanticoagulatory agents. Given that these compounds are active via theoral route, they are used in numerous indications. However, their use isstill limited by certain disadvantages and in particular the risks ofhaemorrhages caused by them and the difficulty of adapting the dosage toa long-term treatment.

Heparins constitute the second category of anticoagulatory agents. Theyare extractive biological substances of the glycosaminoglycan family,which are oligosaccharide compounds with varying chain lengths anddegrees of sulphation. Heparins are used in various types of thromboses,in particular in the treatment or prevention of venous thromboses,optionally combined with other therapies.

The disadvantage of heparins lies in their high anticoagulatory activitywhich may cause haemorrhages, and in their sensitivity to certain serumfactors such as pf4, which requires the use of relatively high doses.

Moreover, heparins are very heterogeneous products. It is thereforedifficult to evaluate their mode of action, to assess the contributionof each of the components in the overall activity of heparin, and,consequently, to increase the antithrombotic activity without increasingthe side effects.

A first solution to the abovementioned disadvantages has been providedby low-molecular weight heparins. These heparins are obtained byfragmentation (depolymerisation) of oligosaccharide chains usingchemical or enzymatic agents. In particular, depolymerisation has beendescribed by a treatment of a heparin ester in the presence of a strongbase (EP 40144). It may also be carried out by treating heparin in thepresence of nitrous acid, or by the action of a heparinase (EP 64452).These various methods lead to mixtures of oligosaccharides having thegeneral structure of the polysaccharides which make up heparin, buthaving a mean molecular weight which is smaller by weight. Moreparticularly, the investigations were directed mainly towardsheparin-derived mixtures having very short oligosaccharide chains. Thus,Patent EP 27089 indicates that heparin-derived oligosaccharide mixturescontaining not more than 8 saccharide units possess an antithromboticspecific activity which is greater than heparin. Similarly,hexasaccharides have been prepared and their antithrombotic propertiesstudied (EP 64452). More recent Patents, EP 84 999 and EP 301 618, onheparin-derived polysaccharides such as hexa-, penta- andtetrasaccharides, may also be mentioned.

However, the products described so far have not enabled the problemsencountered with heparins to be resolved in a completely satisfactorymanner. In particular, it has not been possible to confirm in vivo thecorrelation between the mean molecular mass of the products and theirside effects.

The applicant has now shown that it is possible to obtain, from nativeor depolymerised heparins, oligosaccharide mixtures having greatlyimproved antithrombin properties, and therefore better therapeuticpotential.

In effect, the applicant has shown, unexpectedly, that a substantialpart of the antithrombotic activity of heparin was present in a smalland homogeneous fraction.

The present invention results more particularly from the identificationof monodisperse fractions of heparin having a mean molecular mass ofabout 6 kD and possessing a high antithrombin activity.

As illustrated in the examples, it is therefore possible to obtainmixtures possessing a particularly high antithrombotic activity bycalibrating the molecular mass and by reducing the polydispersity.

One subject of the invention is a mixture of sulphated oligosaccharideshaving the general structure of the constituent oligosaccharides ofheparin, characterised in that it has a mean molecular mass by weight of6±0.6 kD and a polydispersity of about 1, and in that it possesses thecapacity to inhibit the generation of thrombin.

The polydispersity corresponds to the ratio of the mean molecular massof the mixture to its mean molecular weight expressed in numericalterms. It provides information on the molecular homogeneity of themixture. The closer this value is to 1, the more homogeneous is themixture.

In addition to their antithrombin properties, the mixtures of theinvention possess particularly advantageous pharmacokinetic properties.

Consequently, compared with native heparin and its depolymerised forms,the mixtures of the invention exhibit a lower sensitivity to serumfactors such as pf4, which increases their therapeutic potential.

Other advantages of the mixtures of the invention lie in particular inthe reduction of certain undesirable side effects such as:

thrombocytopenic effect. One of the disadvantages of the known mixturesderived from heparin stems from the drop in the number of plateletswhich they can bring about. This undesirable effect is substantiallyreduced when the mixtures of the invention are used,

immunogenic reactions. When such reactions are too intense, it isevident that the therapeutic efficacy of the products is reduced. Theweak immunogenicity of the mixtures of the invention constitutes anotherof their very advantageous pharmacological characteristics.

Furthermore, other advantages of the mixtures of the invention lie inparticular in their excellent plasmatic bioavailability and half-life.

The properties described above permit a particularly effectivepharmacological use especially in the prophylaxis and treatment ofvenous or arterial thromboses. Moreover, they should permit the use ofhigher doses in vivo without increasing the risks of haemorrhage.

In a preferred mode, the mixtures of the invention are more particularlydepolymerised heparin fractions.

As indicated above, the depolymerised heparin may be obtained by anychemical, enzymatic or other technique known to a person skilled in theart, which enable the oligosaccharide chains of heparin to befragmented. In particular, the methods described in Patents EP 40144, EP64452, EP 37319 or EP 337327 are suitable for the invention.

Even more preferably, the mixtures of the invention consist ofoligosaccharides having a 2-O-sulpho-4-enopyranosuronic acid at one oftheir ends.

A particularly advantageous mixture consists of a fraction of heparinwhich is depolymerised by the action of a base on a heparin ester.

The antithrombin activity of the mixtures of the invention may bedemonstrated in a test in which the generation of thrombin is initiatedin the presence of human thromboplastin (extrinsic route) or by contact(intrinsic route). Such a test has been described previously (Hemker etal., Thromb. Haemostas. 56, 9-17, 1986).

This activity may be expressed quantitatively by the amount of productrequired for 25% inhibition of the generation of thrombin. Thus, theincrease in activity of the mixtures of the invention is clearly evidentsince their antithrombin specific activity in vitro is surprisinglyincreased by a factor above 100% compared with the heparin used at thebeginning. Taking into account the particularly advantageouspharmacokinetic properties of the mixtures of the invention, thisincrease in specific activity is even greater in vivo.

More particularly, the mixtures of the invention permit, in a testcarried out on plasma low in platelets, 25% inhibition of the generationof thrombin at concentrations below 300 ng/ml.

Another subject of the invention relates to a method of preparing amixture as defined above, characterised in that a heparin or adepolymerised heparin is fractionated by gel filtration.

The process of the invention brings into play several parameters whosecontrol makes it possible to calibrate the molecular mass of the finalmixture and to determine its polydispersity. These parameters are inparticular the ionic strength of the eluant and the nature of thesupport used.

More preferably, the fractionation is characterised in that the stagesconsisting in (i) dissolving the starting heparin or depolymerisedheparin in the eluant, (ii) passing the solution thus obtained through acolumn at least containing the solid support for the gel filtrationequilibrated beforehand with the same eluant, and (iii) recovering thefractions of the desired molecular weight, are carried out successively.

In a specific embodiment of the invention, the use of a depolymerisedheparin as starting heparin is preferred.

Even more preferably, a heparin depolymerised by the action of a base ona heparin ester is used. In particular, the depolymerisation may becarried out in an aqueous medium or in an inert organic solvent underthe action of an organic or inorganic base such as for example sodium orpotassium hydroxide, an alkali metal carbonate or a tertiary amine(triethylamine, triethylenediamine and the like). The action of the baseon the ester makes it possible to carry out a partial and controlleddepolymerisation of the heparin without modifying its general structure.

More generally, the depolymerisation conditions described in Patent EP40144 may be used in the present invention.

Various types of saline solutions such as solutions of sodium chloridemay be mentioned as eluant which may be used in the method of theinvention. However, the applicant has shown that in order to obtainfractions with the best qualities, it is particularly advantageous tocarry out the fractionation using an eluant chosen from phosphatebuffers such as in particular potassium phosphate, sodium phosphate orNH₄ H₂ PO₄. It is also possible to use NaClO₄ or NH₄ NO₃ solutions whichmake it possible to obtain mixtures with excellent characteristics.

The concentration of the eluant, and therefore its ionic strength, areadjusted to the final mixture desired. In particular, the concentrationof the eluant is advantageously less than 1M and, even more preferably,between 0.1 and 0.5M.

When a phosphate buffer is used, it is particularly advantageous tocarry out the procedure at concentrations of about 0.2M.

In the second stage of the method of the invention, the support used isgenerally chosen as a function of the mean molecular mass of thestarting mixture (native or depolymerised heparin and the like), of thefinal product desired and of the behaviour of the starting mixture inthe eluant used. Advantageously, a polyacrylamide-agarose type gel isused as support. The gels AcA 54, AcA 202, sephadex G-25 or G-50 oralternatively Biogel P30, which give excellent results, may be mentionedby way of example.

In a first particularly advantageous embodiment of the method of theinvention, the solid support is divided among several columns arrangedin series, during the second stage of the fractionation. This variant ofthe invention makes it possible to use substantial final amounts of gelfiltration support without the disadvantages of the prior art, namelythe phenomena of settling essentially. Thus, the separation issubstantially more distinct, including in the high molecular weightrange, in a single fractionation operation, and the supports are moreeasily regenerated.

The number of columns used is adjusted by a person skilled in the art asa function of the volume and the nature of the gel used so as to obtainthe best balance between efficiency of the separation and the adverseeffect due to the settling of the gel.

For practical considerations relating to the implementation, thepreferred number of columns generally used in the second stage of theprocess is less than 20.

By way of illustration, 40 litres of AcA 202 gel may be divided into 104-litre columns.

In another particularly advantageous embodiment of the method of theinvention, at least 2 types of supports having differing separationcharacteristics are used successively in the second stage of thefractionation. This variant of the invention makes it possible to obtaina final fractionation of better quality.

By way of example, the fractionation may be carried out on the followingsequence of gels: AcA 202 - AcA 54 - AcA 202.

For a better implementation of the invention, it is important to usehigh amounts of gel so as to achieve a more distinct separation and toobtain greater homogeneity. However, given the fairly slow flow ratesused for this type of gel filtration, the gel volume should be adaptedto the amount of product to be separated so as to obtain the bestequilibrium between the separation and the effect of longitudinaldiffusion.

Advantageously, in the method of the invention, the starting heparin(g)/gel volume (1) ratio is less than 2, and even more preferablybetween 0.5 and 1.5.

The invention also relates to a method of preparation of weaklydispersed mixtures of oligosaccharides with a molecular weight which iscalibrated by fractionation of heparin or depolymerised heparin by gelfiltration on a solid support, characterised in that the solid supportis divided among several columns arranged in series.

Another subject of the invention relates to a pharmaceutical compositionhaving a mixture as defined above as active ingredient. Such acomposition may be used in a particularly advantageous manner in theprophylaxis or treatment or prevention of thrombotic accidents. Morespecifically, it may be used:

in the prevention of venous thromboses in situations where a riskexists,

in the prevention of arterial thrombotic accidents, especially in thecase of myocardial infarction,

in post-operative regime, in the prevention of venous thromboses insurgical patients, or alternatively,

in the prevention of thromboses in surgical material.

The present invention will be more completely described with the aid ofthe following examples which should be considered as illustrative andnonlimiting.

EXAMPLE 1 Preparation of Mixtures According to the Invention

Depolymerisation of heparin

A solution of 25 g of benzethonium chloride in 125 ml of water is addedto a solution of 10 g of sodium heparinate in 100 ml of water. Theproduct obtained at room temperature is filtered, washed with water andthen dried. 15 g of the benzethonium heparinate thus obtained aredissolved in 75 ml of methylene chloride to which 15 ml of benzylchloride are added. The solution is heated at a temperature of between25 and 35° C. for 25 hours. 90 ml of a 10% solution of sodium acetate inmethanol are then added, filtered, washed with methanol and dried. 10 gof the heparin benzyl ester obtained in the form of a sodium salt underthe conditions described above are dissolved in 250 ml of water. 0.9 gof sodium hydroxide is added to this solution heated to about 60° C. Thetemperature is maintained for I hour 30 minutes at about 60° C. and thereaction mixture is then cooled to around 20° C. and neutralised byadding dilute hydrochloric acid. The mixture is then adjusted to asodium chloride concentration of 10% and the product is precipitated in750 ml of methanol, filtered and dried.

Several glass columns are used for the fractionation:

(a) 1 column with a diameter of 95 mm and a height of 2 m containing 14litres of the AcA 202 gel (gel in the form of polyacrylamide-agarosebeads, with a diameter of between 60 and 140 μm),

(b) 1 column with a diameter of 50 mm and a height of 2 m containing 4litres of the AcA 54 gel (gel in the form of polyacrylamide-agarosebeads, with a diameter of between 60 and 140 μm),

(c) 2 columns with a diameter of 50 mm and a height of 1 m containing 2litres of the AcA 202 gel.

A solution containing 20 g of heparin depolymerised under the conditionsdescribed above is placed at the top of the column (a) and eluted usinga mobile phase consisting of a 0.33M solution of NaCl at a flow rate of210 ml/hour.

The fractions are collected at the outlet of the column (a) and loadedonto the top of the column (b). The elution is carried out with the samesolution and the fractions collected are passed successively through the2 columns (c).

This treatment enables a fraction having the following characteristicsto be separated efficiently and recovered at the outlet of the column(c):

Molecular weight: 6100+/-200

Polydispersity: 1.01

EXAMPLE 2

10 columns with an internal diameter of 2.5 cm and a height of 50 cm,each containing about 0.25 litre of the AcA 202 gel, are connected inseries,

a solution containing 2 g of heparin, which is depolymerised under theconditions of Example 1, is loaded onto the top of the device and elutedusing a 0.2M aqueous solution of KH₂ PO₄ at a flow rate of 0.42 ml/min,

113 fractions of 12.6 ml are collected starting from 21 hours.

The characteristics of these fractions are given in Table 1, in whichthe mean molecular mass was determined by refractometry.

EXAMPLE 3

The procedure is as in Example 2:

10 columns with an internal diameter of 10 cm and a height of 50 cm,each containing 3 to 4 litres of the AcA 202 gel, are connected inseries,

a solution containing 30 g of heparin, which is depolymerised under theconditions of Example 1, is loaded onto the top of the device and elutedusing a 0.2M aqueous solution of KH₂ PO₄ at a flow rate of 6.8 ml/min.

Fractions having the desired polydispersity characteristics areobtained.

EXAMPLE 4

The antithrombin activity of the mixtures of the invention is measuredon plasma stimulated by human thromboplastin (extrinsic route) or bycontact (phospholipids+kaolin: intrinsic route) under the conditionsdescribed above (cf Hemker et al., mentioned above). The activity isestimated by the decrease in the peak of the thrombin generation curverelative to a control carried out in the presence of buffer alone. Theresults are expressed as the IC25: concentration required to obtain 25%inhibition of the generation of thrombin.

Procedure:

1/4 volume of 50 mM tris-HCl buffer, 0.1M NaCl, pH 7.35 with 0.5 mg/mlbovine albumin, containing various concentrations of test samples, isadded to one volume of plasma. After incubating for 5 min at 37° C., thegeneration of thrombin is initiated by the addition of 1/4 volume ofthromboplastin 1:40 diluted in 0.1M CaCl₂ (extrinsic system) or by 6 μMof phospholipids (20% phosphatidylserine, 80% phosphatidylcholine) and0.15 mg/ml of kaolin in 0.1M CaCl₂ (intrinsic system). The generation ofthrombin is obtained by measuring, at regular intervals (15-30 sec), theamydolytic activity on the substrate S2238, a 405 nM chromogenicsubstrate specific for thrombin. Various concentrations of the samplesare tested in order to obtain 25% inhibition of the control.

Results:

On plasma low in platelets

1) extrinsic route

Depolymerised starting heparin: IC25=450 ng/ml

Mixture prepared in Example 1: IC25=200 ng/ml

Activity gain: 125%

2) intrinsic route

Depolymerised starting heparin: IC25=550 ng/ml

Mixture prepared in Example 1: IC25=250 ng/ml

Activity gain: 120%

On plasma high in platelets

Depolymerised starting heparin: IC25=1100 ng/ml

Mixture prepared in Example 1: IC25=500 ng/ml

Under the same conditions, native heparin (nondepolymerised,nonfractionated) possesses no inhibitory activity at 2500 ng/ml.

                  TABLE 1                                                         ______________________________________                                                   MEAN MOLECULAR                                                     FRACTION NO.                                                                             MASS            POLYDISPERSITY                                     ______________________________________                                        14-17      10712           1.027                                              18-20      8400            1.013                                              21-22      7519            1.010                                              23-24      6986            1.011                                              26-27      6365            1.008                                              28-31      5874            1.009                                              32-35      5295            1.011                                              36-40      4761            1.012                                              42-46      4192            1.013                                              48-53      3608            1.016                                              56-61      2988            1.019                                              64-70      2359            1.023                                              75-80      1758            1.029                                              83-85      1476            1.028                                              88-94      1176            1.027                                              ______________________________________                                    

We claim:
 1. A mixture of sulfated oligosaccharides having the structureof constituent oligosaccharides of heparin, that has a mean molecularmass of 6±0.6 kD and a polydispersity of 1 to 1.029, and that possessesa capacity to inhibit the generation of thrombin.
 2. The mixtureaccording to claim 1, wherein the heparin is a depolymerized heparinfraction.
 3. The mixture according to claim 2, consisting of constituentoligosaccharides having a 2-O-sulpho-4-enopyranosuronic acid at one oftheir ends.
 4. The mixture according to claim 3, wherein the heparin isa depolymerized heparin that is depolymerized by the action of a base onan ester of the heparin.
 5. The mixture according to claim 1, having thecapacity, in a test on plasma low in platelets stimulated by humanthromboplastin or by contact, to inhibit the generation of thrombin by25% at concentrations less than 400 ng/ml.
 6. A composition comprisingthe mixture of oligosaccharides according to claim 1 and a buffer.
 7. Amethod of treating or preventing venous or arterial thrombosescomprising administering the mixture according to claim 1 to a patient.8. A method according to claim 7 wherein the arterial thromboses is anarterial thrombotic accident.
 9. The method according to claim 8 whereinthe arterial thrombotic accident is myocardial infarction.
 10. Themethod according to claim 7 wherein the preventing of the venousthromboses is in a post-operative regime on a surgical patient.
 11. Amethod of inhibiting the generation of thrombin comprising administeringthe mixture of oligosaccharides according to claim 1 to a patient.
 12. Amethod of preventing thromboses in a surgical material comprisingcontacting the material with the mixture of claim 1.